Chromatography is used to separate mixtures of substances into their individual components. The basic principles of chromatography involve a stationary phase (usually a solid or a liquid supported on a solid) and a mobile phase (usually a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Since different components travel at different rates, the components separate from each other during the process and can be isolated.
In column chromatography, stationary phase particles are packed into a column and the mobile phase is allowed to flow through the stationary phase. This method relies on gravity to force the mobile phase through the stationary phase. During column chromatography, the smaller the stationary phase particle size, the greater the separation of components. However, smaller particles have greater resistance to flow.
HPLC is an improved form of column chromatography. Instead of a solvent being allowed to drip through a column under gravity, it is forced through the column under high pressures of up to 400 atmospheres, thereby increasing the speed of the process and allowing the use of smaller stationary phase particles. Thus, HPLC allows for improved separation of the components of the mixture.
FIG. 1 is a schematic of a conventional HPLC system 100. A sample mixture 120 is introduced to system 100 through injector valve 130. Injector valve 130 allows sample 120 to be introduced into HPLC loop 140 with any excess sample 120 directed to waste 110. Once sample 120 is in HPLC loop 140, injector valve 130 is activated to allow mobile phase 160 to be pumped into system 100 via pump 150. Pump 150 forces mobile phase 160 through injector valve 130 and HPLC loop 140 and into column 170. Column 170 is filled with a stationary phase 175. As mobile phase 160 is forced through injector valve 130, HPLC loop 140, and column 170, the individual components of sample 120 are separated out. As the separated components flow out of the column, detector 180 analyzes the results.
One common method of detecting substances that have passed through the column uses ultra-violet absorption. Many organic compounds absorb UV light of various wavelengths. Therefore a beam of UV light is directed through the stream of liquid coming out of the column and detected on the opposite side of the stream. The amount of light absorbed will depend on the amount of a particular compound that is passing through the beam at the time. Based on the output of the detector, it is possible to identify the separated components of the sample.
Conventional HPLC is often employed as a method of analysis of different components in a given sample. In the production of radiolabeled pharmaceuticals, the radiolabeled pharmaceutical synthesis step is often followed by a purification step. HPLC is one purification method to isolate the radiolabeled pharmaceutical from the synthesis mixture. In a radiolabeled pharmaceutical HPLC purification system, a radiation detector is used along with the UV detector to identify the components of the sample. The detection of a peak in the UV or radiation detector signal indicates the presence of the radiolabeled pharmaceutical. A valve following the detectors is installed to direct fluid flow to either a waste vial or to a product collection vial. Once the UV chromatogram peak or the radiation chromatogram peak of the desired radiolabeled pharmaceutical appears, the valve is switched to collect this product.
As mentioned above, the first step of the HPLC process is to load a specific amount of the sample to be purified/separated into a fixed volume HPLC loop. The other end of the loop is directed to waste to catch any waste that overflows from the loop. In a regular HPLC setup, the sample is injected into the loop with a syringe and overflows to waste are common. However, in the production of radiolabeled pharmaceuticals, it is desirable to avoid overflows to waste, thereby minimizing process loss. Further complicating the process is that the task of loading the loop is often automated.
One efficient way of transporting liquid is by using a push gas to push liquid through a series of tubes. The push gas has to be turned off at the right time to avoid sending the crude radiolabeled product to waste. Prior systems use a bubble detector or a liquid detector to determine when the radiolabeled product has been completely loaded into the HPLC loop and to stop the push gas. However, these systems are unreliable.
Therefore, it is desirable to have a reliable device to automatically limit the amount of sample that is injected into the HPLC loop.